Osteoporosis, or porous bone, is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased risk of fractures of the hip, spine (vertebras), and wrist. Wrongly often thought of as an “old woman's disease”, osteoporosis affects not only one in three postmenopausal women, but also one in five men over the age of 50, younger women and even children. Osteoporosis is known as the “silent epidemic” because there are often no symptoms until a fracture occurs. Once a fracture has occurred, the risk of a future fracture is at least doubled within one year.
People with broken bones suffer severe pain and disability, resulting in a loss of quality of life and independence. There is also an increased risk of death—24% of women and 33% of men die within one year after a hip fracture. Yet the sad reality in Europe is that osteoporosis is usually not diagnosed in time. Despite the availability of approved diagnostic and therapeutic options, people continue to suffer fractures, many of which, could have been prevented. However, this disease could be prevented and treated if an accurate method could properly prognose low Bone Mineral Density and fractures.
As Europe's population ages and becomes more sedentary, the number of people affected by osteoporosis will increase significantly—hip fractures alone are expected to double in the next 50 years. Unless osteoporosis prevention and treatment becomes a priority for government and health care providers, this growing number of osteoporotic fractures will have a serious impact on society, not just in terms of people's quality of life, but also in regard to increased expenditure for health care, rehabilitation and nursing care.
Hip fractures are a serious result of osteoporosis. These fractures almost invariably result in chronic pain, reduced mobility, disability, and loss of independence. Hip fractures also result in mortality rates of up to 20-24% in the first year after a hip fracture. Various studies have shown that loss of function and independence among survivors is profound, with ca. 40% unable to walk independently and ca. 60% requiring assistance a year later. Because of these losses, around a third of the patients are totally dependent or in a nursing home in the year following a hip fracture. The number and cost of hip fractures are commonly used to measure the burden of osteoporosis because, unlike other osteoporotic fractures, statistics for hip fracture are usually available through hospital records. Vertebral fractures and wrist fractures often go unrecorded and, in the case of vertebral fractures, often remain undiagnosed.
People with osteoporosis suffer from a reduction in their bone mass and bone quality—put simply, their bones become fragile, leading to an increased risk of fractures. Bone density loss is without noticeable symptoms. The most reliable way to determine loss of bone mass is to have a bone mineral density (BMD) test. DXA scans (Dual Energy X-Ray Absorptiometry) are the most accurate and most commonly used method of BMD measurement and are usually used to measure spine and hip bone densities. DXA scans are vital in order to properly diagnose and monitor osteoporosis. Yet access to bone mineral density measurement is sub-optimal in many European countries. Reasons include limited availability of densitometers, restrictions in personnel permitted to perform scans, low awareness of the usefulness of BMD testing, limited or non-existent reimbursement. Many of the DXA scanners are not available to the public health care system, or regional disparities mean that some parts of a country are under-serviced.
Therefore, there remains in the art, a need for reliable means of predicting fractures and/or low bone mineral density. Early diagnosis or prognosis could affect therapeutic decisions.
A comprehensive osteoporosis treatment program includes a focus on proper nutrition, exercise, and safety issues to prevent falls that may result in fractures. In addition, there are therapeutic medications to slow or stop bone loss, increase bone density, and reduce fracture risk. Currently treatments for Osteoporosis include alendronate, raloxifene, risedronate, and ibandronate and they are approved by the U.S. Food and Drug Administration (FDA) for preventing and treating postmenopausal osteoporosis. Teriparatide is approved for treating the disease in postmenopausal women and men at high risk for fracture. Estrogen/hormone therapy (ET/HT) is approved for preventing postmenopausal osteoporosis, and calcitonin is approved for treatment. In addition, alendronate is approved for treating osteoporosis in men, and both alendronate and risedronate are approved for use by men and women with glucocorticoid-induced osteoporosis.
Studies have identified a number of genes in which one or more genetic variations result in a higher or lower risk of contracting osteoporosis, a better or worse response to drugs and furthermore, a better or worse prognosis of the disease and the risk for suffering fractures.
However, there remains a need for a reliable means of characterising such genetic variations which can be used for clinical purposes.
DNA chips are often used to determine alleles at generic loci.
In 2001, the Consortium for the Human Genome Project and the private company Celera presented the first complete example of the human genome with 30,000 genes. From this moment on, the possibility of studying the complete genome or large scale (high-throughput) studies began. So-called “DNA-chips”, also named “micro-arrays”, “DNA-arrays” or “DNA bio-chips” are apparatus that functional genomics can use for large scale studies. Functional genomics studies changes in the expression of genes due to environmental factors and to genetic characteristics of an individual. Gene sequences present small interindividual variations at one unique nucleotide called an SNP (“single nucleotide polymorphism”), which in a small percentage are involved in changes in the expression and/or function of genes that cause certain pathologies. The majority of studies which apply DNA-chips study gene expression, although chips are also used in the detection of SNPs.
The first DNA-chip was the “Southern blot” where labelled nucleic acid molecules were used to examine nucleic acid molecules attached to a solid support. The support was typically a nylon membrane.
Two breakthroughs marked the definitive beginning of DNA-chip. The use of a solid non-porous support, such as glass, enabled miniaturisation of arrays thereby allowing a large number of individual probe features to be incorporated onto the surface of the support at a density of >1,000 probes per cm2. The adaptation of semiconductor photolithographic techniques enabled the production of DNA-chips containing more than 400,000 different oligonucleotides in a region of approximately 20 μm2, so-called high density DNA-chips.
In general, a DNA-chip comprises a solid support, which contains hundreds of fragments of sequences of different genes represented in the form of DNA, cDNA or fixed oligonucleotides, attached to the solid surface in fixed positions. The supports are generally glass slides for the microscope, nylon membranes or silicon “chips”. It is important that the nucleotide sequences or probes are attached to the support in fixed positions as the robotized localisation of each probe determines the gene whose expression is being measured. DNA-chips can be classified as:                high density DNA-chips: the oligonucleotides found on the surface of the support, e.g. glass slides, have been synthesized “in situ”, by a method called photolithography.        low density DNA-chips: the oligonucleotides, cDNA or PCR amplification fragments are deposited in the form of nanodrops on the surface of the support, e.g. glass, by means of a robot that prints those DNA sequences on the support. There are very few examples of low density DNA-chips which exist: a DNA-chip to detect 5 mutations in the tyrosinase gene; a DNA-chip to detect mutations in p53 and k-ras; a DNA-chip to detect 12 mutations which cause hypertrophic cardiomypathy; a DNA-chip for genotyping of Escherichia coli strains; or DNA-chips to detect pathogens such as Cryptosporidium parvum or rotavirus.        
For genetic expression studies, probes deposited on the solid surface, e.g. glass, are hybridized to cDNAs synthesized from mRNAs extracted from a given sample. In general the cDNA has been labelled with a fluorophore. The larger the number of cDNA molecules joined to their complementary sequence in the DNA-chip, the greater the intensity of the fluorescent signal detected, typically measured with a laser. This measure is therefore a reflection of the number of mRNA molecules in the analyzed sample and consequently, a reflection of the level of expression of each gene represented in the DNA-chip.
Gene expression DNA-chips typically also contain probes for detection of expression of control genes, often referred to as “house-keeping genes”, which allow experimental results to be standardized and multiple experiments to be compared in a quantitative manner. With the DNA-chip, the levels of expression of hundreds or thousands of genes in one cell can be determined in one single experiment. cDNA of a test sample and that of a control sample can be labelled with two different fluorophores so that the same DNA-chip can be used to study differences in gene expression.
DNA-chips for detection of genetic polymorphisms, changes or mutations (in general, genetic variations) in the DNA sequence, comprise a solid surface, typically glass, on which a high number of genetic sequences are deposited (the probes), complementary to the genetic variations to be studied. Using standard robotic printers to apply probes to the array a high density of individual probe features can be obtained, for example probe densities of 600 features per cm2 or more can be typically achieved. The positioning of probes on an array is precisely controlled by the printing device (robot, inkjet printer, photolithographic mask etc) and probes are aligned in a grid. The organisation of probes on the array facilitates the subsequent identification of specific probe-target interactions. Additionally it is common, but not necessary to divide the array features into smaller sectors, also grid-shaped, that are subsequently referred to as sub-arrays. Sub-arrays typically comprise 32 individual probe features although lower (e.g. 16) or higher (e.g. 64 or more) features can comprise each subarray.
One strategy used to detect genetic variations involves hybridization to sequences which specifically recognize the normal and the mutant allele in a fragment of DNA derived from a test sample. Typically, the fragment has been amplified, e.g. by using the polymerase chain reaction (PCR), and labelled e.g. with a fluorescent molecule. A laser can be used to detect bound labelled fragments on the chip and thus an individual who is homozygous for the normal allele can be specifically distinguished from heterozygous individuals (in the case of autosomal dominant conditions then these individuals are referred to as carriers) or those who are homozygous for the mutant allele.
Another strategy to detect genetic variations comprises carrying out an amplification reaction or extension reaction on the DNA-chip itself.
For differential hybridisation based methods there are a number of methods for analysing hybridization data for genotyping:                Increase in hybridization level: The hybridization level of complementary probes to the normal and mutant alleles are compared.        Decrease in hybridization level: Differences in the sequence between a control sample and a test sample can be identified by a fall in the hybridization level of the totally complementary oligonucleotides with a reference sequence. A complete loss is produced in mutant homozygous individuals while there is only 50% loss in heterozygotes. In DNA-chips for examining all the bases of a sequence of “n” nucleotides (“oligonucleotide”) of length in both strands, a minimum of “2n” oligonucleotides that overlap with the previous oligonucleotide in all the sequence except in the nucleotide are necessary. Typically the size of the oligonucleotides is about 25 nucleotides. The increased number of oligonucleotides used to reconstruct the sequence reduces errors derived from fluctuation of the hybridization level. However, the exact change in sequence cannot be identified with this method; sequencing is later necessary in order to identify the mutation.        
Where amplification or extension is carried out on the DNA-chip itself, three methods are presented by way of example:
In the Minisequencing strategy, a mutation specific primer is fixed on the slide and after an extension reaction with fluorescent dideoxynucleotides, the image of the DNA-chip is captured with a scanner.
In the Primer extension strategy, two oligonucleotides are designed for detection of the wild type and mutant sequences respectively. The extension reaction is subsequently carried out with one fluorescently labelled nucleotide and the remaining nucleotides unlabelled. In either case the starting material can be either an RNA sample or a DNA product amplified by PCR.
In the Tag arrays strategy, an extension reaction is carried out in solution with specific primers, which carry a determined 5′ sequence or “tag”. The use of DNA-chips with oligonucleotides complementary to these sequences or “tags” allows the capture of the resultant products of the extension. Examples of this include the high density DNA-chip “Flex-flex” (Affymetrix).
For genetic diagnosis, simplicity must be taken into account. The need for amplification and purification reactions presents disadvantages for the on-chip extension/amplification methods compared to the differential hybridization based methods.
Typically, DNA-chip analysis is carried out using differential hybridization techniques. However, differential hybridization does not produce as high specificity or sensitivity as methods associated with amplification on glass slides. For this reason the development of mathematical algorithms, which increase specificity and sensitivity of the hybridization methodology, are needed (Cutler D J, Zwick M E, Carrasquillo M N, Yohn C T, Tobi K P, Kashuk C, Mathews D J, Shah N, Eichler E E, Warrington J A, Chakravarti A. Genome Research; 11:1913-1925 (2001).
Thus, despite advances in technology, the problems of existing methods is simultaneously analysing a large number of genetic variations in a sensitive, specific and reproducible way, has prevented the application of DNA-chips for routine use in clinical diagnosis